Neural circuits are the fundamental functional units of the nervous system. A basic understanding of circuit function will provide an important basis for understanding how these circuits malfunction in neurological disorders. The study of neural circuits in small and relatively simple model animals such as C. elegans and Drosophila has many advantages, including genetic manipulability and amenability to optical techniques. Circuit analysis in these organisms has been buoyed by the recent development of 'optogenetic' methods for stimulating and inhibiting neural activity using light-sensitive ion channels and pumps [1]. Progress in optogenetics requires not only development and optimization of new opsin molecules but also new strategies and technologies for perturbing specific opsin-expressing neurons. In this project, we will develop optical and genetic methods for manipulating neural circuits with single- neuron resolution in freely moving C. elegans. This project extends previous work by Dr. Fang-Yen, in which machine vision algorithms and lasers patterned by a digital micromirror device (DMD) were used to achieve spatiotemporal control of neural activity in freely behaving worms [2]. This earlier system was limited to a spatial resolution of about 20-30 microns, which is insufficient to selectively illuminate single neurons in the animal's nerve ring (brain). In this project we will develop a next-generation system capable of resolving single neurons and subcellular features. We will approach this goal in three directions. First, we will develop instrumentation and machine vision algorithms to automatically image and track individual neurons and processes using fluorescence imaging. We will use a dual-magnification optical system to simultaneously track behavior of the entire worm and fluorescence in a smaller region. Second, we will design and implement predictive algorithms to illuminate tracked targets with compensation for the latency due to image processing and data transfer. This system will be designed with real-time feedback such that fine-tuning of its parameters can be done in an automated manner. Third, we will use our system, in combination with other methods, to elucidate the mechanisms of modulation of locomotory behaviors by dopaminergic and serotonergic circuits. By enabling, for the first time, the dynamic perturbation of individual or multiple neurons in a behaving animal, the technology we develop will become an important tool for the analysis of neural circuits, with numerous advantages compared with existing methods. In addition to improving our understanding of the circuit basis of behavior, these studies will help provide a circuit-level context for interpreting genetic mutants, for example in C. elegans models of synaptic transmission, neuronal development, and neurodegeneration. While the focus of this project is on C. elegans, we expect that our methods will be readily extensible to other model organisms. This project will be centered in Dr. Fang-Yen's laboratory but will draw on the expertise of several unpaid consultants at the University of Pennsylvania or nearby. These include Dr. David Raizen (Dept. of Neurology), an expert in C. elegans genetics and behavior, Dr. Brian Chow (Dept. of Bioengineering), an expert on optogenetic reagents, and Dr. Niels Ringstad (New York University), an expert in C. elegans genetics and neurotransmitter signaling.